Cancer treatment using c-type natriuretic peptide

ABSTRACT

The present invention includes a method of utilizing four peptide hormones to inhibit the growth of cancer(s). A dramatic decrease in the number of human pancreatic adenocarcinoma cells (i.e., the type of cancer with the highest mortality, with patients only surviving four months) was observed responsive to treatment. The application of the invention would be to utilize one or more of these peptide hormones alone and/or in combination to treat cancer. The ability of these peptide hormones to decrease the number of adenocarcinoma cells has implications for adenocarcinomas at other sites in the body with the majority of cancers of the breast, colon and prostate also being adenocarcinomas. Adenocarcinomas also occur in the lung and other tissues. Treatment of a wide variety of cancers in addition to adenocarcinomas is anticipated by the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of co-pending U.S.application Ser. No. 10/908,604, filed May 18, 2005; which applicationis a Continuation in Part of U.S. patent application Ser. No., entitled,“10/708,688”, filed Mar. 18, 2004; which application claims priority ofU.S. Provisional Application No. 60/320,018, filed Mar. 19, 2003.

BACKGROUND OF THE INVENTION

Cancer of the prostate is the most commonly diagnosed neoplasm in menwith over 232,000 new cases estimated in the United States in 2005,i.e., 30% of all new cancer diagnoses in men. Mortality from prostatecancer remains a significant problem with current treatment(s) with anestimated 30,350 deaths from prostate cancer in 2005 making it thesecond leading cause of cancer death in men after lung cancer.

Lung cancer is the leading cause of cancer death in both men and womenin the United States, and this same trend is seen in many othercountries. In the United States in 2004 it was estimated that lungcancer accounted for 31% of cancer deaths in men and 25% of cancerdeaths in women. Small-cell lung cancers account for 20% to 25% of alllung cancers. Small-cell lung cancer differs from other lung cancers inthat it metastasizes very early and can rarely be cured surgically. Inthe current management of small-cell lung cancers the majority ofpatients are treated first with chemotherapy plus radiotherapy, but withthis combination survival is only 20% at 3 years. There is an increasedincidence of secondary cancers with the cisplatin- and cytoxan-basedregimens used to treat small-cell lung cancer, and the chemotherapeuticregimens for small-cell lung cancer containing doxorubicin may inducecardiac myopathy. All the presently utilized chemotherapeutic agents fortreatment of small-cell lung cancers have some side-effects, includingmyelosuppression. Atrial natriuretic peptides consist of a family ofpeptides that are synthesized by three different genes then stored asthree different prohormones [i.e., 126 amino acid (a.a.) atrialnatriuretic peptide (ANP), 108 a.a. brain natriuretic peptide (BNP), and103 a.a. C-type natriuretic peptide (CNP) prohormones] (8-10). Withinthe 126 a.a. ANP prohormone are four peptide hormones i.e., long actingnatriuretic peptide (LANP), vessel dilator, kaliuretic peptide, andatrial natriuretic peptide (ANP), whose main known biologic propertiesare blood pressure regulation and maintenance of plasma volume inanimals and humans. The BNP and CNP genes, on the other hand, appear toeach synthesize only one peptide hormone within their respectiveprohormones, i.e., BNP and CNP. Each of these peptide hormonescirculates in healthy humans with vessel dilator and LANP concentrationsbeing 17- to 22-fold higher than ANP, 33- to 48-fold higher than BNP and124 to 177-fold higher than CNP.

Therefore, what is needed is a method of treating cancer that can bothimprove survival, lacks the side effects of current therapies, andultimately decrease morbidity.

SUMMARY OF INVENTION

In a general embodiment, the present invention comprises a method ofinhibiting the growth of cancer cells comprising the step of contactingat least one target cell with an effective amount of a peptide hormonederived from the C-natriuretic peptide (CNP) prohormone. The peptidehormone administered is derived from the CNP prohormone. In oneembodiment, the target cell is chosen from the group consisting ofadenocarcinomas, small cell carcinomas and squamous cell carcinoma, andthe peptide hormone is administered in vivo.

In another embodiment, the invention includes a method of inhibiting thegrowth of cancer cells comprising the step of co-administering, to atleast one target cell, an effective amount of a combination of peptidehormones derived from the ANP prohormone and a peptide hormone derivedfrom the C-natriuretic peptide (CNP) prohormone. The combination ofpeptide hormones is derived from the ANP prohormone and a peptidehormone derived from the C-natriuretic peptide (CNP) prohormone isselected from the group consisting of atrial natriuretic peptide, longacting natriuretic peptide, vessel dilator, kiliuretic peptide, andC-natriuretic peptide. In another embodiment the target cell is chosenfrom the group consisting of adenocarcinomas, small cell carcinomas andsquamous cell carcinoma, and the combination of peptide hormones isadministered in vivo.

The present investigation is a new therapeutic approach that has none ofthe side effects of current cancer therapeutic agents. This approach isto utilize peptide hormones made mainly in the atria of the heart calledatrial natriuretic peptides. These peptide hormones have recently beenfound to have significant anticancer effects on human breast andpancreatic adenocarcinomas as well as small-cell and squamous cell lungcarcinomas.

The present investigation incorporates the atrial peptides made by allthree genes within the heart, i.e., the above four peptide hormones plusBNP and CNP. When the four peptide hormones from the ANP gene were foundin dose-response studies to cause a significant decrease in the numberof human prostate adenocarcinoma cells (i.e., up to 97.4% within 24hours), it was investigated whether the mechanism(s) of this decrease inthe number of human prostate adenocarcinoma and small-cell carcinomacells and the ability of these peptides to inhibit further proliferationof these cancer cells after their decreased number was owing toinhibition of DNA synthesis. It was then determined whether theirintracellular mediator cyclic GMP could reproduce their effects on humanprostate adenocarcinoma cells and DNA synthesis. The inventors furtherexamined the prostate adenocarcinoma cells to determine if they havenatriuretic peptide (NPR)-A and -C receptors to mediate these peptidehormones effects since natriuretic peptide receptors have never beendemonstrated on prostate cancer cells.

In the present invention, these cardiovascular peptide hormones decreasethe number of human small-cell lung cancer cells showing that thesepeptides are a good addition to the therapeutic regimen for small-celllung cancers, as they do not cause myleosuppression, secondary tumours,cardiac myopathy or any known side-effect of cancer chemotherapeuticagents. Thus, their use allows for the current anticancerchemotherapeutic agents to be used in a lower dose(s) to obtain the sameeffect. Utilizing one or more of these peptide hormones might also allowfor a dose-escalation of currently used chemotherapeutic agents as animportant strategy for overcoming drug resistance. Thus, thisdose-escalation is less than the present escalation of currentchemotherapeutic agents, as the peptide hormones of the currentinvestigation would decrease the number of cancer cells as demonstratedin the present invention before this dose-escalation would begin. Withless cells to kill, as the vessel dilator kills 92% of the cells within24 hours, this dose escalation does not have to increase as much as incurrent clinical trials, thereby achieving higher complete remissionrates and increasing cures with less side-effects, as the total dose(s)of the chemotherapeutic agents to achieve cure is less if the abovepeptide hormones (without current anticancer agents' side-effects) aregiven first.

These peptide hormones, which circulate normally in the human body, haveno known cytoxic effects on normal cells and only one known side-effect.This side-effect, i.e. hypotension, has only been observed with ANP andBNP and never with the vessel dilator, LANP, or kaliuretic peptide.These last three peptide hormones with the best safety profile alsodecrease adenocarcinoma cells in vitro the most, small-cell lung cancercells in vitro the most (present investigation), and decreaseadenocarcinoma tumour volume in vivo the most out of the ANPs.Presently, utilized chemotherapy very commonly causes toxicity in theform of nausea, vomiting, alopecia, and myelosuppression. None of thesetoxicities occur with the ANP hormones.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1. Decrease in human prostate adenocarcinoma cells after 24 hourexposure to 1 μM of long acting natriuretic peptide (LANP), vesseldilator, kaliuretic peptide, atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP) and C-type natriuretic peptide (CNP).

FIGS. 2A-2D are graphs showing the specific decrease in human prostatecancer cell number with 1 μM concentrations of these peptide hormones.

FIG. 3. Dose-response of vessel dilator (VD), long acting natriureticpeptide (LANP), kaliuretic peptide (KP) and atrial natriuretic peptide(ANP)'s anticancer effects on human prostate adenocarcinoma cells.

FIG. 4. Specific decrease in DNA synthesis by vessel dilator, longacting natriuretic peptide (LANP), kaliuretic peptide, and atrialnatriuretic peptide (ANP).

FIG. 5. Natriuretic peptide receptors (NPR) A- and C-receptors arepresent in human prostate cancer cells.

FIG. 6 Is a graph showing the decrease in human small-cell lung cancercells after a 24-h exposure to 1 mM of long acting natriuretic peptide(LANP), vessel dilator, kaliuretic peptide, atrial natriuretic peptide(ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide(CNP).

FIG. 7 Time course of decrease in human small-cell lung cancer cellnumber with 1 mM of the vessel dilator (□), longacting natriureticpeptide (O, LANP), kaliuretic peptide (Δ, atrial natriuretic peptide(ANP, ▴), brain natriuretic peptide (BNP, ▪), C-type natriuretic peptide(⋄), respectively, and cyclic GMP (cGMP, 1 μM, ♦) compared withplacebo-treated () small-cell lung cancer cells.

FIG. 8 Evaluation of brain natriuretic peptide (BNP) and C-typenatriuretic peptide (CNP) at 10-fold and 100-fold higher concentrationsthan the concentration (1 μM) that caused a significant decrease inhuman small-cell lung cancer cell number with the other fourcardiovascular hormones.

FIG. 9 Dose-response curves of the vessel dilator on the number of humansmall-cell lung cancer cells.

FIG. 10 Sustained 92% or greater decrease in human small-cell lungcancer cell number compared with the untreated (control) cancer cellsover 4 days secondary to the 100-μM concentration of the vessel dilator.

FIG. 11 is a graph demonstrating inhibition of DNA synthesis by thevessel dilator, long-acting natriuretic peptide (LANP), kaliureticpeptide, and atrial natriuretic peptide (ANP) in human small-cell lungcancer cells.

FIG. 12 Western blot of natriuretic peptide receptors (NPRs) A- and C-are present in human small-cell lung cancer cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

The present invention is the first evaluation of whether prostateadenocarcinomas cells contain natriuretic peptide receptors. Both theNPR-A and NPR-C receptors were present in human prostate adenocarcinomacells. This knowledge helps to explain brain natriuretic peptide (BNP)and C-natriuretic peptide (CNP)'s decreased biologic effects in thesecancer cells at their 1 μM concentrations. ANP binds to both the NPR-Aand C-receptors with a higher affinity than BNP or CNP. The binding tothe NPR-A receptor is ANP>BNP>>CNP while binding to the NPR-C receptoris ANP>CNP>BNP. Thus, one needs significantly more of BNP and CNP tocause the same effects observed with ANP and that is why they have noeffects when compared at the same concentration where ANP first hasanticancer effects.

Although prostate cancer is curable with surgery if it is detected andremoved at an early stage, unfortunately is not always detected when itis surgically curable. With current treatment with anti-androgens, etc.it is still the second leading cause of cancer death in men after lungcancer. With an estimated 30,350 prostate cancer deaths expected tooccur this year (2005) with surgery and current cancer chemotherapy plusanti-androgens there is an urgent need to develop new approaches totherapy of prostate cancer. The present investigation details not onlyone but four new potential therapies which kill up to 97.4% of humanprostate cancer cells within 24 hours. These peptide hormones whichcirculate normally in the human body have no known cytoxic effects tonormal cells and only one known side effect. This side effect, i.e.,hypotension, has only been observed with ANP and BNP and never withvessel dilator, LANP, or kaliuretic peptide in human or animal subjects.These last three peptide hormones with the best safety profile alsodecrease breast and pancreatic adenocarcinoma cells in vitro the mostand decrease adenocarcinoma tumor volume in vivo the most of the atrialnatriuretic peptides. There was no hypotension in the animals wherethese peptide hormones decreased up to 50% of the tumor volume of humanpancreatic adenocarcinomas in vivo in one week. Presently utilizedchemotherapy commonly causes toxicity in the form of nausea, vomiting,alopecia, and myelosuppression. None of these toxicities occur with theatrial natriuretic peptide hormones.

Example I Prostate Adenocarcinoma Cells

All four of the peptide hormones synthesized by ANP gene, i.e., LANP,vessel dilator, kaliuretic peptide and ANP had very significant(P<0.001) effects at their 1 mM concentrations decreasing the number ofcancer cells by 87% to 97.4% within 24 hours.

Vessel dilator was the most potent of these peptide hormones indecreasing the number of the human prostate cancer cells at each of therespective concentrations of these peptide hormones (FIG. 3). Vesseldilator had significant (P<0.001) effects within 24 hours at its 1 μMconcentration (62% decrease in number of prostate adenocarcinoma cells)and inhibited any further proliferation of the prostate cancer cellsfrom 24 to 96 hours (P<0.001; FIG. 2A). In the dose-response curves ofthe present investigation, when vessel dilator concentration wasincreased 10-fold and 100-fold (i.e., 10 and 100 μM), vessel dilatordecreased the number of human cancer cells 72% and 91% within 24 hours(FIG. 3). When vessel dilator concentration was increased to 1 mM, 97.4%of the cancer cells were killed in 24 hours i.e., only three cancercells (3±2.74 cells) that had not been killed (FIG. 3). Thus, vesselnearly eliminated all of the human cancer cells within 24 hours. Inseveral of the fields there were no cancer cells at all, i.e., vesseldilator had killed 100% of the cancer cells within these fields in 24hours. Vessel dilator also decreased human pancreatic and breastadenocarcinomas in vitro the most and small-cell and squamous cell lungcancer cells in vitro the most and decreased human pancreaticadenocarcinoma tumor volume the most in vivo suggesting that it has themost significant anticancer properties of these peptide hormones.

The other three peptide hormones synthesized by the ANP gene effects ondecreasing the number of prostate adenocarcinoma cells were significant,however. When the concentrations of LANP, kaliuretic peptide, and ANPwere increased in dose-response curves to 1 mM, they caused a verysignificant 87% to 89% decrease in the number of human prostateadenocarcinoma cells within 24 hours. There appears to be a differencein these peptide hormones' ability to decrease cancer cell numberdepending upon the type of cancer. Kaliuretic peptide (1 μM), forexample, ability to decrease the number of prostate adenocarcinoma cellsat 24 hours (30% decrease) although similar to its effect (30%) onsmall-cell lung cancer cells was not as good (37% decrease) as withhuman pancreatic adenocarcinoma cells. It should be noted that there wasno decrease in the number of cells when examined immediately afteraddition of the respective peptide hormones indicating that the dataobtained was not due to artifact. It is also important to note thatafter 24 hours of incubation with the four peptide hormones thatcellular debris was present suggesting that cellular necrosis wasoccurring.

The effect of these four peptide hormones on decreasing the number ofhuman pancreatic cancer cells was very specific as evidenced by theantibody studies where the addition of the respective antibodies to thepeptide hormones resulted in a maximum of only 2% of the cancer cellsbeing decreased (FIG. 2). This maximum 2% was seen only with thekaliuretic peptide antibody plus kaliuretic peptide and only at one timeperiod with no decrease whatsoever at 72 and 96 hours with kaliureticpeptide plus its antibody. With each of the other three antibodies plustheir respective peptide the decrease was 0 to 1% which is significantly(P<0.0001) different than the peptide alone where significant decreasesoccurred. Since the decrease in cancer cell number secondary to thesepeptide hormones could be completely blocked by their respectiveantibodies this indicates that the peptides (rather than some otherfactor) were specifically the cause of the decrease in human prostatecancer cell number. The respective antibodies alone caused a slight butinsignificant increase in cancer cell number (FIG. 2). The antibodies,thus, would not be useful to treat prostate adenocarcinomas as opposedto the data of the present investigation demonstrating an 87% to 97.4%decrease in cancer cell number within 24 hours with the four peptidehormones themselves.

Each of the four peptide hormones from the ANP prohormone inhibited 68%to 89% of the amount of DNA synthesis in the human prostateadenocarcinoma cells. The present investigation demonstrated that theDNA synthesis-inhibiting properties of these peptide hormones weredirectly due to the peptide hormones themselves as when their specificantibodies were incubated with the peptide hormones the antibodiescompletely blocked these four peptide hormones ability to decreasecancer cell DNA synthesis (FIG. 4). The antibodies by themselves (FIG.4) did not significantly block DNA synthesis. These findings suggestthat one important mechanism of action of these peptide hormones toinhibit cancer cell number and their proliferation is via their abilityto inhibit DNA synthesis. This finding is similar to the ability ofthese peptide hormones to inhibit DNA synthesis in human pancreaticadenocarcinoma cells and human breast adenocarcinoma cells.

With respect to the mechanism of how these peptide hormones inhibit DNAsynthesis, one of the second messengers of their biologic effects, i.e.,cyclic GMP was found using 8-bromo cyclic GMP (1 mM) to inhibit DNAsynthesis 87% in the human prostate cancer cells. Cyclic GMP's mimickingthe effects of these peptide hormones on DNA synthesis in the same cellssuggests that cyclic GMP is one of the mediators of these peptidehormones' ability to inhibit DNA synthesis in prostate adenocarcinomacells. Further evidence that the cyclic GMP inhibition of DNA synthesisin prostate cancer cells may be important for these peptide hormonesanticancer growth effects is that when cyclic GMP was infusedsubcutaneously for a week in athymic mice with human pancreaticadenocarcinomas, it inhibited 95% of the growth of the human pancreaticadenocarcinoma compared to placebo-treated adenocarcinomas.

The cell line (ATCC number CRL-1435; PC-3) of human prostateadenocarcinoma cells was derived in 1979 by M. E. Kaighn et al. from agrade IV prostate adenocarcinoma from of a 62 year old Caucasian man.These cells, when injected into athymic mice, form tumors within 21days.

Culture of the Prostate Adenocarcinoma Cells

Propagation of these cells was in Ham's F1K medium with 2 mM/L-glutamineadjusted with addition of 1.5 g/L sodium bicarbonate, 90%; andheat-inactivated 10% fetal bovine serum at a temperature of 37° C. asrecommended by the ATCC. Cells were dispensed into new flasks withsubculturing every 6-8 days. The growth medium was changed every threedays.

After the prostate adenocarcinoma cells were subcultured for 24 hoursthey were then seeded to coverslips in 24 well plates with 1 mL of theabove Ham's F12K media. After 24 hours, wells were washed twice withphosphate buffered saline to remove the fetal bovine serum. Removal ofserum was done to completely remove all variables (EGF, etc.) present inserum in order that interpretation of any data obtained would bestraightforward. After 24 hours of serum deprivation, media volume wasreduced to 250 μL per well with or without the respective peptidehormones in dose response curves with concentrations up to and including1 mM (1% of this volume). Human prostate adenocarcinoma cells were thenincubated for various periods of time. The number of prostateadenocarcinoma cells were then counted with a cell counter evaluatingten fields of the microscope slide at 40× along the X-axis. Thisevaluation was repeated on six separate occasions with the number ofprostate adenocarcinoma cells reflecting 60 observations for each group,i.e., 60 observations for controls and 60 observations for each of thesix groups with respective peptide hormones.

Specificity of These Peptide Hormones to Decrease the Number of HumanProstate Adenocarcinoma Cells.

To determine if the decrease in number of human pancreaticadenocarcinoma cells was specific, these peptide hormones (1 μM) andtheir specific antibodies (5 μM) were incubated together for 24, 48, 72,and 96 hours. Both the peptide hormones and their antibodies were fromPhoenix Pharmaceuticals, Inc., Belmont, Calif. The antibodies (5 μM)were also incubated alone (i.e., without the peptide hormones) at eachof the above time points.

Determination of DNA Synthesis.

To investigate whether these peptide hormones were inhibiting DNAsynthesis, bromodeoxyuridine (BrdU) incorporation into the prostateadenocarcinoma cells was utilized. DNA synthesis and doubling of thegenome take place during the synthetic or S phase (32,33).Bromodeoxyuridine is a thymidine analog incorporated into nuclear DNAduring the S phase of the cell cycle. After 24 hours in culture with 1μM of LANP, vessel dilator, kaliuretic peptide, ANP, BNP, or CNP,respectively, or with no peptide hormone (i.e., control), BrdU in afinal concentration of 10 μM in the cell culture medium was added for 45minutes -which is the time in which the cells are in the logarithmicphase of cell proliferation. For immunohistochemistry, a BrdU in situdetection kit was utilized.

The incorporation of the BrdU stain into the nucleus was counted using aNikon Inverted Diaphot-TMD Microscope (Tokyo, Japan). The number ofstained nuclei were compared in the six peptide hormone groups to thepositive control group. To investigate DNA synthesis, BrdU incorporationby immunochemistry has been demonstrated to be equally good as³H-thymidine incorporation and has the advantage that it provides highresolution.

Cyclic GMP Effects on DNA Synthesis.

Cyclic GMP is one of the known mediators of the previously describedbiologic effects of these peptide hormones. All four of these peptidessynthesized by the ANP gene-induced vasodilations of vasculature aremediated by increased cyclic GMP concentrations via enhancing guanylatecyclase activity. The inventors have previously shown that each of thesepeptide hormones increase cyclic GMP while simultaneously dilatingvasculature. 8 bromo-cyclic GMP reproduces these vasodilatory effects.For the present investigation of part of the mechanism of these peptidehormones' ability to inhibit DNA synthesis in prostate adenocarcinomacells, 8-bromoguanosine 3′,5′-cyclic monophosphate was utilized. 8-bromocyclic GMP is a cell-permeable analog of cyclic GMP.

ANP Receptors in Human Prostate Adenocarcinoma Cells.

Prostate cancers have never been examined to determine if they havenatriuretic receptors. When it was found that these ANPs decreased thenumber of human prostate adenocarcinoma cells, it was then evaluatedwhether prostate cancer cells have ANP receptors to mediate theseeffects. Western blots of the natriuretic peptide receptors (NPR) A- andC- were performed as follows:

Western Blotting. Seventy-five micrograms of protein extract from humanprostate adenocarcinoma cells, measured by using the bicinchonic acid(BCA) protein assay kit was loaded onto each lane of a Criterion Precast7.5% Tris-HCl gel, separated by electrophoresis (200 volts for 60 min),and then transblotted onto a nitrocellulose membrane for 75 min at 100volts in Towbin buffer. Blots were blocked for 1 hour at roomtemperature in a 5% solution of dry milk, washed×3 with Tris bufferedsaline, and then incubated for 1 hour in a 5% solution of bovine serumalbumin in Tris-buffered saline that contained a 1:4,000 dilution ofA035 polyclonal antibody directed against the COOH terminus of the NPR-Areceptor protein or containing Tris-buffered saline with a 1:1,000dilution of antibody to the NPR-C receptor. After being washed×4 withTris-buffered saline, the membranes were incubated for 1 hour at roomtemperature in a solution of dry milk with a 1:6,000 and 1:3,000dilutions of goat anti-rabbit IgG antibody for NPR-A and NPR-Creceptors, respectively. After three washings with Tris-buffered saline,the bands were identified by enhanced chemiluminescence reagents andvisualized in a luminescent image analyzer. Specificity was revealed bythe presence of a signal in rat lung (positive control) and absence of asignal with normal rabbit serum, rabbit IgG, and after preabsorption ofthe NPR-A antibody with NPR-A protein or preabsorption of the NPR-Cantibody with NPR-C protein. Monoclonal anti-B-actin antibody was usedas a loading control.

Decrease in Number of Human Prostate Adenocarcinoma Cells by FourPeptide Hormones Synthesized by the ANP Gene.

The number of prostate adenocarcinoma cells after 24 hours without theaddition of any of the peptide hormones averaged 89±2 cells perhigh-powered field when ten fields of the coverslip were evaluated at×40 along the x-axis. This evaluation was repeated on six separateoccasions with the above number reflecting sixty observations of thenumber of control prostate adenocarcinoma cells and sixty observationsof each of the six groups with addition of one of the peptide hormones(FIG. 1). The decrease in prostate cancer cells was significant atP<0.001 with vessel dilator, P<0.01 with ANP, and P<0.05 with kaliureticpeptide and LANP when evaluated by repeated analysis of variance(ANOVA). There was no significant decrease in prostate cancer cellnumber secondary to BNP or CNP when evaluated by ANOVA. The decrease innumber of human prostate cancer cells secondary to vessel dilator wassignificantly greater (P<0.05) than the decrease secondary to any of theother natriuretic peptides when evaluated by ANOVA. Cyclic GMP's (cGMP)(1 μM) decrease in prostate adenocarcinoma cells was significant atP<0.05 when evaluated by ANOVA.

The addition of 1 μM of long acting natriuretic peptide (LANP) for 24hours decreased the number of human prostate cancer cells to 60±3 i.e.,a 32% decrease (P<0.05) in the number of prostate adenocarcinoma cellswith the LANP (FIG. 1). Vessel dilator at 1 μM for 24 hours had an evenmore dramatic decrease (62%; P<0.001) in the number of the humanprostate adenocarcinoma cells (FIG. 1). Vessel dilator decreased thenumber of lung cancer cells from 89±2 cells to 33±3. Kaliuretic peptideat 1 μM for 24 hours decreased the number of human prostateadenocarcinoma cells 30% (P<0.05), i.e., to 62±3 prostate cancer cells(FIG. 1).

The number of human prostate adenocarcinoma cells in culture decreased37% (P<0.05) when exposed to atrial natriuretic peptide (1 μM) for 24hours (FIG. 1). Brain natriuretic peptide and CNP, each at 1 μM, onlydecreased the number of human prostate adenocarcinoma cells 0.8% and 1%,respectively, after 24 hours of incubation (not significant). Thus, withrespect to their ability to inhibit the growth of human prostate cancercells when these cells were exposed to identical concentrations of thesesix peptide hormones for 24 hours, vessel dilator>ANP>kaliureticpeptide>LANP>CNP>BNP. When the number of cells was examined immediatelyafter the incubation of the respective peptide hormones within thecells, there was not any decrease in the number of cancer cells. In thewells with decreased number of cells secondary to the cardiac hormones,there was evidence of cellular debris.

Specificity of the Ability of These Peptide Hormones to Decrease theNumber of Human Pancreatic Adenocarcinoma Cells.

To determine if the significant effects of these peptide hormones todecrease the number of human prostate adenocarcinoma cells were specificthe inventors utilized these peptides hormones specific antibodies in a1:5 concentration of peptide hormone to their respective antibody. Asshown in FIGS. (2A) vessel dilator, (2B) long acting natriuretic peptide(LANP), (2C) kaliuretic peptide, and (2D) atrial natriuretic peptide(ANP)'s anticancer effects compared to placebo-treated human prostateadenocarcinoma cells at 24, 48, 72, and 96 hours were significant atP<0.001 compared to placebo when evaluated by repeated analysis ofvariance (ANOVA). The addition of their respective specific antibodies(5 μM) blocked these peptide hormones' anticancer effects which wassignificant at P<0.001 when evaluated by repeated measures of ANOVA at24, 48, 72, and 96 hours of incubation. The addition of the respectiveantibody alone resulted in a slight increase in cancer cell number thatwas not significant when evaluated by ANOVA.

When these peptide hormones (each at 1 μM) were incubated with theirspecific antibodies (5 μM) the decrease in cancer cell number secondaryto vessel dilator alone of 62% was reduced to 1% only (FIG. 2A). Therewas also only a 1% decrease in cell number with LANP plus its antibody(FIG. 2B). Kaliuretic peptide plus its antibody resulted in a 0.4%decrease versus a 30% decrease with kaliuretic peptide alone (FIG. 2C).These antibodies studies also indicated that ANP's effects were specificwith the 37% decrease in cell number with ANP alone decreased to 2% whenits antibody was added (FIG. 2D). The addition of specific antibodyblocked each of these peptides ability to decrease cancer cells atP<0.0001.

When these specificity experiments were extended to 48, 72, and 96 hoursof incubation of antibody plus peptide hormones, for vessel dilator plusantibody (FIG. 2A) the decrease in number of cancer cells was 1%, 0%,and 1%, respectively (P<0.0001). LANP plus its antibody resulted in 0%,1%, 1% decrease in cancer cells (FIG. 2B) at 48, 72, and 96 hours. Withkaliuretic peptide plus its antibody there was a 0%, 0%, and 1% decreasein prostate cancer cell number at 48, 72, and 96 hours, respectively(FIG. 2C). When ANP's antibody plus ANP were incubated for 48, 72, and96 hours there was a 1%, 0%, and 0% decrease in prostate cancer cells(FIG. 2D).

When the antibodies alone (same concentration, i.e., 5 μM) wereincubated for 24 hours without the addition of any of the peptidehormones, the vessel dilator antibody resulted in a 5% increase (ratherthan decrease) in prostate cancer cell number while the ANP antibodyresulted in a 3% increase in prostate adenocarcinoma cells. With theLANP and kaliuretic peptide antibodies alone for 24 hours there was a0.1% decrease and 2% increase, respectively. Thus, there was nosignificant decrease in cancer number with the antibodies alone, butrather 3 of the 4 antibodies caused an increase in cancer cell numberwithin 24 hours. When the antibody alone experiments were extended for48, 72, and 96 hours there was a 3% increase in cancer cells at eachtime period with vessel dilator (FIG. 2A) with a 0%, 1% increase, and 1%decrease respectively at these time periods with LANP (FIG. 2B). Whenthe kaliuretic peptide antibody alone was incubated for 48, 72, and 96hours there was a 2%, 1%, and 1% increase, respectively (FIG. 2C), whilewith ANP antibody alone there was a 2%, 1%, and 0% increase at thesetime points (FIG. 2D).

Decreased Cellular Proliferation after Initial Decrease in HumanProstate Adenocarcinoma Cell Number.

When the prostate adenocarcinoma cells were exposed for longer periodsof time e.g., 48, 72, and 96 hours to vessel dilator, LANP, kaliureticpeptide, ANP, BNP, and CNP each at 1 μM, there was a inhibition ofproliferation of the prostate cancer cells after the decrease in thenumber of these cancer cells at 24 hours by the peptide hormones fromthe ANP gene (FIG. 2). Thus, when exposed to vessel dilator, LANP,kaliuretic peptide and ANP for 48 hours the inhibition of the number ofcancer cells compared to untreated human prostate adenocarcinoma cellswas 63% (P<0.001), 32%, 31% and 38% (P<0.05 for these three peptides),respectively (FIG. 2). At 72 hours and 96 hours, the decrease in numberof prostate adenocarcinoma cells secondary to vessel dilator was 63% atboth time periods (P<0.001, FIG. 2). At both 72 and 96 hours, the numberof prostate adenocarcinoma cells was reduced 33% by LANP (P<0.05 forboth) compared to untreated prostate adenocarcinoma cells at these timeperiods (FIG. 2B). At 72 and 96 hours, the number of cancer cells withkaliuretic peptide present was decreased by 31% and 33%, respectively,(P<0.05 for each) (FIG. 2C). The number of human prostate cancer cellsboth at 72 and 96 hours decreased 38% secondary to ANP (P<0.05 for both)(FIG. 2D). Thus, proliferation was inhibited by these peptide hormonesfor three days after the initial decrease in cell number in the first 24hours (FIG. 2). There was no significant decrease in human prostateadenocarcinoma cancer number secondary to BNP or CNP (each at 1 μM) at48, 72, or 96 hours. Thus, at 48, 72, and 96 hours the decrease inprostate cancer cells was 1%, 1%, and 1%, respectively with BNP whilethe decrease secondary to CNP was 2%, 1.5%, and 1%, respectively.

Dose-Response Studies.

Dose response studies utilizing 10, 100 and 1000-fold higherconcentrations for 24 hours revealed that with each increase in theconcentrations of the four peptide hormones synthesized by the ANP genethere was a further decrease (P<0.05) the number of prostate cancercells (FIG. 3). At each increasing concentration of these four peptidehormones there was a significantly (P<0.05) increased decrease in numberin prostate cancer cells when evaluated by repeated measures of ANOVA.At their 1 mM concentration these peptide hormones decreased 87 to 98%of the prostate cancer cells (P<0.001 compared to control) whenevaluated by repeated measures of ANOVA. Vessel dilator caused the samedecrease as the other peptide hormones at a 10-fold lower concentrationas observed in FIG. 3.

Vessel dilator which decreased the number of human prostateadenocarcinoma cells 63% at 1 μM further decreased the number of cancer72% at 10 μM, 91% at 100 μM and 97.4% at 1 mM when incubated for 24hours (FIG. 3). Thus, vessel dilator at 1 mM eliminated almost all ofthe human prostate adenocarcinoma cells within 24 hours (i.e., therewere only 3±2.24 (5D) cells left with several of the fields that wereexamined having no cancer cells whatsoever still alive). Long actingnatriuretic peptide (LANP) which decreased 32% of the prostate cancercells at its 1 μM concentration decreased the number of cancer cells57%, 69%, and 87% at its 10 μM, 100 μM, and 1 mM concentrations,respectively (FIG. 3). Kaliuretic peptide caused a 54%, 71%, and 88%decrease in prostate cancer cell number when its 10 μM, 100 μM, and 1 mMconcentrations. ANP decreased the number of prostate cancer cells 55%,70%, and 89% at its 10 μM, 100 μM, and 1 mM concentrations (FIG. 3).Thus, at their 1 mM concentrations these peptide hormones decreased 87to 98% of the prostate cancer cells within 24 hours (FIG. 3).

Cyclic GMP Decreases Prostate Adenocarcinoma Cell Number.

Cyclic GMP at 1 μM decreased the number of human prostate cancer cells33% at 24 hours (FIG. 1). The decrease in number of human prostateadenocarcinoma cells at 48, 72, and 96 hours was 35% at each timeperiod, respectively, with 1 μM of cyclic GMP (P<0.05 at all time pointscompared to control). Dose response curves with cyclic GMP revealed thatat 10 μM, 100 μM, and 1 mM of 8-bromo cyclic GMP the number of prostatecancer cells decreased 57%, 66%, and 84%, respectively.

Inhibition of DNA Synthesis by Four Peptide Hormones.

To help determine the mechanism of the prostate adenocarcinoma cells'decrease in number and decreased cellular proliferation by the abovefour peptide hormones, the inventors next investigated whether theireffects were due to an inhibition of DNA synthesis as they have beendemonstrated to decrease DNA synthesis in pancreatic and breastadenocarcinoma cells. Vessel dilator, LANP, kaliuretic peptide and ANPeach at their 1 μM concentrations inhibited DNA synthesis when incubatedwith human prostate cancer cells for 24 hours by 89%, 68%, 76% and 79%,respectively (P<0.001 for each) (FIG. 4). There was not any significantdecrease in DNA synthesis in the prostate cancer cells secondary to BNPor CNP at 1 μM (i.e., 4 and 5% decrease) (FIG. 4).

The 68 to 89% decrease in DNA synthesis secondary to these four peptidehormones (FIG. 4) (each at 1 μM) was significant (P<0.001) compared tocontrol (i.e., untreated) cells and compared to DNA synthesis when thepeptide hormones specific antibodies (5 μM) were added to the peptidehormones (P<0.001) when evaluated by repeated measures of analysis ofvariance (ANOVA). These peptide hormones' antibody alone did notsignificantly affect DNA synthesis when evaluated by repeated measuresof ANOVA.

To determine if the decreases in DNA synthesis were specifically due tothe respective peptide hormones of this investigation, the peptidehormones (each at 1 μM) plus their respective antibodies (5 μM) wereinvestigated for their ability to inhibit DNA synthesis. When vesseldilator antibody was added with vessel dilator there was only a 1%decrease in DNA synthesis versus an 89% decrease in DNA synthesis withvessel dilator alone (FIG. 4). The vessel dilator antibody alone caused0% decrease in DNA synthesis (FIG. 4). The LANP antibody plus LANPresulted in a 6% decrease in DNA synthesis while the LANP antibody alonecaused a 1% decrease in DNA synthesis (FIG. 4). Kaliuretic peptideantibody plus kaliuretic peptide decreased DNA only 5% versus a 76%decrease in DNA synthesis with kaliuretic peptide alone (P<0.01). Thekaliuretic peptide antibody alone caused no (0%) decrease in DNAsynthesis (FIG. 4). ANP plus ANP antibody resulted in a 4% decrease inDNA synthesis versus a 79% decrease in DNA synthesis with ANP alone.When the ANP antibody was utilized by itself there was a 5% decrease inDNA synthesis (FIG. 4). These peptide hormones effects on DNA synthesisare, thus, very specific and definitely related to the peptide hormonesthemselves.

Cyclic GMP Inhibits DNA Synthesis in Human Prostate Cancer Cells.

To help define the mechanism(s) for these peptide hormone's ability todecrease DNA synthesis, one of the known mediators of these peptidesbiologic effects, i.e., cyclic GMP was investigated to determine if itcould inhibit DNA synthesis in these same prostate cancer cells. 8-bromocyclic GMP decreased DNA synthesis in prostate adenocarcinoma cells by56% (P<0.01) at its 1 μM concentration (FIG. 4).

NPR-A and -C Receptors are Present in Human Prostate Cancer Cells.

Prostate adenocarcinoma cancer cells have never been evaluated todetermine whether they have NPR-A and/or -C receptors. When the humanprostate adenocarcinoma cells were evaluated by Western blots, the NPRA-and C-receptors were demonstrated to be present (FIG. 5).

Western blot analysis with 1:4,000 dilution of A035 polyclonal antibodydirected against the COOH terminus of the natriuretic peptide A-receptor(NPR-A) and 1:1,000 dilution of antibody to the NPR-C receptor. Theupper graph demonstrates the positive rat lung control (PANC) for theNPR-A receptor and NPR-A receptor in the human prostate adenocarcinomacells (PC-3). The lower graph demonstrates the NPR-C receptor at 66kiloDaltons (kDa) in the human prostate cancer cells as well as in thepositive control (left panel). Beta-galactosidase (130 kDa) and BSA (70kDa) were used in addition to BIO RAD Precision Plus Protein Dual colorstandards to identify the bands corresponding to the NPR-A and NPR-Creceptors, respectively. Re-probing with Beta-Actin was used as aloading control.

Example II Small-Cell Lung Cells

This invention is the first evidence that vessel dilator, ANP, LANP,kaliuretic peptide and CNP can decrease the number of human small-celllung carcinoma cells. All four of the peptide hormones synthesized bythe ANP gene, i.e. LANP, vessel dilator, kaliuretic peptide and ANP havebeen previously investigated by the inventors for their effects onadenocarcinomas, i.e. human pancreatic and breast adenocarcinomas.Similar results were found with human pancreatic and breastadenocarcinomas to each of the four peptide hormones synthesized by theANP gene decreasing the number of human pancreatic adenocarcinoma cellsduring the first 24 hours 34% to 65% at their 1-μM concentrations withsimilar decreases in human breast adenocarcinoma cells. Thus, these fourpeptide hormones significantly decrease the number of cancer cells inboth adenocarcinomas and small-cell lung carcinomas. The presentinvention indicates therefore that these four peptide hormonessignificantly decrease the number of cancer cells of at least twodifferent types of cancers within 24 hours. This information, plus theknowledge that one of these peptide hormones, i.e. ANP, decreases thenumber of hepatoblastoma cells and proliferation in neuroblastoma cellsin culture, suggests that these peptide hormones possibly havegeneralized anticancer effects, i.e. have the ability to decrease thenumber of cancer cells from a variety of different cancers. The abilityof these four peptide hormones to completely stop the growth of humanpancreatic adenocarcinomas in vivo with three of the four peptidehormones decreasing the volume of this human cancer up to 50% in 1 week,highlights the clinical cancer treatment relevance of these peptidehormones.

Brain natriuretic peptide and CNP at their 1-μM and 10-μM concentrationsdid not have similar effect on the number of human small-cell lungcancer cells. Brain natriuretic peptide did not have any significantanticancer effects, even when its concentration was 100-fold higher thanthe concentration of the four peptide hormones synthesized by the ANPprohormone gene, which significantly decreased the human small-cellcancer cell number. C-natriuretic peptide did have significant effectswhen its concentration was increased to 100-fold higher than theconcentration where the four peptide hormones from the ANP prohormonegene had significant effects (39% decrease).

Vessel dilator was the most potent of these peptide hormones indecreasing the number of the small-cell lung cancer cells. Vesseldilator had significant (P<0.001) effects within 24 hours (63% decreasein number of small-cell lung cancer cells) and inhibited any furtherproliferation of the small-cell lung cancer cells from 24 to 96 hours(P<0.001; FIG. 7). In the dose-response curves of the presentinvestigation, when vessel dilator concentrations were increased 10-foldand 100-fold (i.e. 10 and 100 μM), it decreased the number of humancancer cells 73% and 92% within 24 hours (FIG. 9) with no proliferationof these cancer cells in the 3 days that followed (FIG. 10). After 3days with 100 μM of vessel dilator, only six human cancer cells (6±0.5cells) had not been killed. Thus, the vessel nearly eliminated all ofthe human cancer cells within 24 hours. Vessel dilator also decreasedhuman pancreatic adenocarcinomas in vitro the most and decreased humanpancreatic adenocarcinoma tumour volume the most in vivo, demonstratingthat it has the most significant anticancer properties of these peptidehormones.

The other three peptide hormones synthesized by the ANP gene effects ondecreasing the number of small-cell lung carcinoma cells weresignificant, however. Atrial natriuretic peptide (1 μM) decreased thenumber of small-cell carcinoma cells at 24 hours (39% decrease), whichis more than its ability to decrease the number of pancreaticadenocarcinoma cells at 24 hours, i.e. 34% . On the other hand,kaliuretic peptide's (1 μM) ability to decrease the number of small-celllung carcinoma cells at 24 hours was not as good with small-cell lungcarcinoma cells (30%) as with human pancreatic adenocarcinoma cells(37%). Thus, there appears to be a difference in these peptide hormones'ability to decrease the cancer cell number depending upon the type ofcancer.

When the four peptide hormones that had significant effects at 1 μM wereadded together (each at 1 μM) for 24 hours they decreased the number ofhuman small-cell lung cancer cells by 62%, which was similar to theability of the vessel dilator to decrease the number of cancer cells(i.e. 63%) by itself. It should be noted that there was no decrease inthe number of cells when examined immediately after addition of therespective peptide hormones, indicating that the data obtained was notowing to artifact. It is also important to note that cellular debris waspresent after 24 hours of incubation with the four ANP prohormonepeptides, suggesting that cellular necrosis was occurring.

Each of the four peptide hormones from the ANP prohormone inhibited 68%to 82% of the amount of DNA synthesis in these small-cell lung carcinomacells. These findings suggest that one important mechanism of action ofthese peptide hormones to inhibit cancer cell number and theirproliferation is via their ability to inhibit DNA synthesis.

With respect to the mechanism of how these peptide hormones inhibit DNAsynthesis, one of the second messengers of their biologic effects, i.e.cyclic GMP, was found using 8-bromo cyclic GMP to inhibit DNA synthesisup to 50% in the small-cell lung carcinoma cells. Cyclic GMP's mimickingof the effects of these peptide hormones on DNA synthesis in the samecells suggests that cyclic GMP is one of the mediators of these peptidehormones' ability to inhibit DNA synthesis in small-cell lung cancercells. The concentrations of cyclic GMP that inhibited DNA synthesis inthe small-cell lung carcinoma cells are identical to the concentrationsof cyclic GMP measured within tissues secondary to these peptidehormones. Further evidence that the cyclic GMP inhibition of DNAsynthesis in small-cell lung cancer cells may be important for thesepeptide hormones anticancer growth effects is that whencyclic GMP wasinfused subcutaneously for a week in athymic mice with human pancreaticadenocarcinomas, it inhibited 95% of the growth of the human pancreaticadenocarcinoma compared with placebo-treated adenocarcinomas.

Both the NPR-A and NPR-C (i.e. clearance) receptors were present inthese small-cell lung cancer cells. This knowledge helps to explainBNP's and CNP's lack of biologic effects in these cancer cells at their1-μM concentrations. Atrial natriuretic peptide binds to both the NPR-Aand C-receptors with a higher affinity than BNP or CNP. Binding to theNPR-A receptor is ANP>BNP>>CNP while binding to the NPR-C receptor isANP>CNP>BNP. Binding of human BNP to the human NPRC receptor is an orderof magnitude lower than ANP, indicating that an order of magnitudelarger concentration of BNP is necessary to be present for BNP to haveeffects. In the present investigation the inventors have investigatedBNP and CNP, which had no effects at 1 μM whereas the other peptidehormones did, at 10-fold and 100-fold higher concentrations. At 10-foldhigher (i.e. 10 μM) concentrations of BNP and CNP, neither significantlydecreased the number of human small-cell lung cancer cells. Anticancereffects were observed at the 100-fold higher concentration of CNP, buteven at the 100-fold higher concentration no any anticancer propertieswere found with BNP. The ability of CNP to have effects at 100 μM butnot at 100-fold lower concentrations that the other four cardiovascularhormones had effects while BNP still did not have any effect when itsconcentration was increased 100-fold may relate in part to the abilityof these peptide hormones to bind to the NPR-C receptor which maymediate some of these peptides biologic effects as described below.Binding to the NPR-C receptor is ANP>CNP<BNP. Thus, ANP, which bindsmore avidly to this receptor, had effects at 100-fold lowerconcentrations than CNP, which is next in affinity for this receptor,while BNP, which has the lowest affinity for this receptor, had noanticancer effects. As the NPR-A receptor is thought to be the activereceptor mediating ANP, BNP and CNP effects, part of the anticancereffects found with ANP may be mediated through this receptor found to bepresent in the small-cell lung cancer cells.

The NPR-C receptor was demonstrated to be present for the first time inthe present invention in human small-cell lung cancer cells. Althoughthe NPR-C receptor (i.e. clearance receptor) has been thought to mainlyclear ANP, BNP, and CNP from the circulation, as it does not haveguanylate cyclase or protein kinase attached to it, as with the NPR-Areceptor, to produce cyclic GMP which is the main mediator of ANP, BNP,and CNP effects, there is evidence now that ANP may signal via the NPR-Creceptor in smooth muscle cells. In smooth muscle cells, ANP afterbinding to the NPR-C receptor initiates a signal cascade consisting of aCa2+ influx, and activation of endothelial nitric oxide synthase withresulting formation of nitric oxide activating cytosolic guanylatecyclase, which in turn increases the concentration of the intracellularmediator cyclic GMP. This would be a mechanism of increasing cyclic GMPwithout utilizing the NPR-A receptor to help mediate the cyclic GMPeffects of decreasing smallcell lung cancer cell number and DNAsynthesis found in the present investigation.

Small-Cell Lung Cancer Cells

A cell line (ATCC number CRL-2195, SHP-77) was derived in 1977 by E. R.Fisher, A. Palekar and J. D. Paulson from a nonencapsulated primary lungtumour from the apical portion of the upper lobe of the left lung of a54-year-old Caucasian man. These cells, when injected into athymic mice,form tumours with a doubling time of 96 hours.

Culture of the Small-Cell Lung Carcinoma Cells

Propagation of these cells was in Roswell Park Memorial Institute (RPMI)1640 medium with 2 mM L⁻¹-glutamine adjusted with addition of 1.5 g L⁻¹sodium bicarbonate, 4.5 g L⁻¹ glucose, 10 mM HEPES, 1 mM of 90% sodiumpyruvate and heat-inactivated 10% fetal bovine serum at a temperature of37° C., as recommended by the ATCC. Cells were dispensed into new flaskswith subculturing every 6-8 days. The growth medium was changed every 3days.

Research Protocol

The research protocol for the small-cell lung carcinoma cells wassimilar to that of the of the prostate adenocarcinoma cells discussedabove. The protocol varied in that After subculturing for 24 hours, thesmall-cell lung carcinoma cells were then seeded to coverslips in24-well plates with 1 mL of the abovementioned RPMI media (rather thanHam's F12k media as discussed above).

Determination of DNA Synthesis

To investigate whether these peptide hormones were inhibiting DNAsynthesis, bromodeoxyuridine (BrdU) incorporation into the small-celllung carcinoma cells was utilized. Bromodeoxyuridine was from BDBioscience, San Jose, Calif. DNA synthesis and doubling of the genometake place during the synthetic or S phase. Bromodeoxyuridine is athymidine analogue incorporated into nuclear DNA during the S phase ofthe cell cycle. After 24 hours in culture with 1 μM of LANP, vesseldilator, kaliuretic peptide, ANP, BNP, or CNP, respectively, or with nopeptide hormone (i.e. control), BrdU in a final concentration of 10 μMin the cell culture medium was added for 45 min: the time in which thecells are in the logarithmic phase of cell proliferation. Forimmunohistochemistry, a BrdU in situ detection kit was utilized.Incorporation of the BrdU stain into the nucleus was counted using aNikon Inverted Diaphot-TMD Microscope. The number of stained nuclei werecompared in the six peptide hormone groups to the positive controlgroup. BrdU incorporation by immunochemistry has been demonstrated to beequally good as ³H-thymidine incorporation, with the advantage ofproviding high resolution.

ANP Receptors in Small-Cell Lung Carcinoma Cells

On finding that these ANPs decreased the number of human small-cellcarcinoma cells, it was then evaluated whether small-cell lungcarcinomas have ANP receptors to mediate these effects. Western blots ofthe natriuretic peptide receptors (NPRs) A- and C- were performed asfollows.

Western Blotting

Seventy-five micrograms of protein extract from small-cell lungcarcinoma cells, measured using the bicinchonic acid (BCA) protein assaykit, was loaded onto each lane of a Criterion Precast 7.5% Tris-HCl gel,separated by electrophoresis (200 V for 60 min), and then transblottedonto a nitrocellulose membrane Hybond-C Extra, for 75 min at 100 V inTowbin buffer. Blots were blocked for 1 hours at room temperature in a5% solution of dry milk, washed×3 with Trisbuffered saline, and thenincubated for 1 hours in a 5% solution of bovine serum albumin inTris-buffered saline containing a 1:4000 dilution of A035 polyclonalantibody directed against the COOH terminus of the NPR-A receptorprotein or containing Tris-buffered saline with a 1:1000 dilution ofantibody to the NPR-C receptor. After being washed×4 with Tris-bufferedsaline, the membranes were incubated for 1 hours at room temperature ina solution of dry milk with a 1:6000 and 1:3000 dilutions of goatantirabbit IgG antibody for the NPR-A and NPR-C receptors, respectively.After three washings with Tris-buffered saline, the bands wereidentified by enhanced chemiluminescence reagents and visualized in aluminescent image analyzer. Specificity was revealed by the presence ofa signal in rat lung (positive control) and absence of a signal withnormal rabbit serum, rabbit IgG, and after preabsorption of the NPR-Aantibody with NPR-A protein or preabsorption of the NPRC antibody withNPR-C protein. Monoclonal anti-B-actin antibody (Sigma) was used as aloading control.

Decrease in number of small-cell carcinoma cells by four peptidehormones synthesized by the ANP gene.

The number of small-cell lung carcinoma cells after 24 hours without theaddition of any of the peptide hormones averaged 86±9 cells perhigh-powered field when 10 fields of the coverslip were evaluated at ×40along the X-axis. This evaluation was repeated on six separate occasionswith the above number reflecting 60 observations of the number ofcontrol small-cell lung carcinoma cells and 60 observations of each ofthe six groups with addition of one of the cardiac hormones (FIG. 6).The addition of 1 μM of LANP for 24 hours decreased the number of humansmall-cell lung carcinoma cells to 68±6, i.e. a 21% decrease (P<0.05) inthe number of small-cell carcinoma cells with the LANP (FIG. 6). Vesseldilator at 1 μM for 24 hours had an even more dramatic decrease (63%;P<0.001) in the number of the human small-cell lung carcinoma cells(FIG. 6). Vessel dilator decreased the number of lung cancer cells from86±9 cells to 32±4. Kaliuretic peptide at 1 μM for 24 hours decreasedthe number of human small-cell lung carcinoma cells 30% (P<0.05), i.e.to 60±6 small-cell lung cancer cells (FIG. 6).

The decrease in small-cell lung cancer cells was significant at P<0.001with the vessel dilator, P<0.01 with ANP, and P<0.05 with kaliureticpeptide and LANP when evaluated by repeated analysis of variance(ANOVA). There was no significant decrease in small-cell lung cancercell number secondary to BNP or CNP when evaluated by ANOVA. Thedecrease in number of small-cell lung cancer cells secondary to thevessel dilator was significantly greater (P<0.05) than the decreasesecondary to any of the other natriuretic peptides when evaluated byANOVA. Cyclic GMP's (cGMP) (1 μM) decrease in small-cell lung cancercells was significant at P<0.05 when evaluated by ANOVA. Combination ofthe four peptide hormones that had significant effects, i.e. vesseldilator, LANP, kaliuretic and ANP (each at 1 μM), caused a verysignificant (P<0.001) decrease in the number of human small-cell lungcancer cells, but this decrease was no more significant than that causedby the vessel dilator alone when evaluated by ANOVA.

The number of human small-cell lung cancer cells in culture decreased39% (P<0.05) when exposed to ANP 1 μM for 24 hours (FIG. 6). Brainnatriuretic peptide and CNP, each at 1 μM, only decreased the number ofsmall-cell lung carcinoma cells 0% and 4%, respectively, after 24 hoursof incubation (not significant). Thus, with respect to their ability toinhibit the growth of human small-cell carcinoma cells when these cellswere exposed to identical concentrations of these six peptide hormonesfor 24 hours, vessel dilator>ANP>kaliuretic peptide>LANP>CNP>BNP. Addingthe four peptide hormones which had significant effects together (eachat 1 μM) resulted in a 62% decrease (i.e. similar to vessel dilatoralone) in human small-cell cancer cell number (FIG. 6). When the numberof cells was examined immediately after the incubation of the respectivepeptide hormones within the cells, there was no decrease in the numberof cancer cells. In the wells with decreased number of cells secondaryto the cardiac hormones, there was evidence of cellular debris.

Decreased Cellular Proliferation after Initial Decrease in Small-CellLung Carcinoma Cell Number

When small-cell lung cancer cells were exposed for longer periods oftime, e.g. 48, 72, and 96 hours to the vessel dilator, LANP, kaliureticpeptide, ANP, BNP, and CNP each at 1 μM, there was a inhibition ofproliferation of the small-cell lung carcinoma cells after the decreasein the number of these cancer cells at 24 hours by the peptide hormonesfrom the ANP gene (FIG. 7). Thus, when exposed to the vessel dilator,LANP, kaliuretic peptide and ANP for 48 hours, the inhibition of thenumber of cancer cells compared with untreated small-cell carcinomacancer cells was 54% (P<0.001), 20%, 21% and 24% (P<0.05 for these threepeptides), respectively. At 72 hours and 96 hours, the decrease innumber of small-cell carcinoma cells secondary to the vessel dilator was59% and 58% (P<0.001, FIG. 7). At both 72 and 96 hours, the number ofsmall-cell carcinoma cells was reduced 21% by LANP (P<0.05 for both)compared with untreated small-cell lung cancer cells at these timeperiods. At 72 and 96 hours, the number of cancer cells with kaliureticpeptide present was decreased by 28% and 27%, respectively, comparedwith untreated small-cell lung carcinoma cells (P<0.05 for each) (FIG.7). The number of small-cell lung cancer cells at 72 and 96 hoursdecreased secondary to ANP 30% and 32% (P<0.05 for both) compared withthe number of small-cell lung cancer cells at these same time periodswithout the addition of any peptide hormone (FIG. 7). Thus,proliferation was inhibited by these cardiac peptide hormones for 3 daysafter the initial decrease in cell number in the first 24 hours (FIG.7). There was no significant increase in proliferation of any of thecancer cells when exposed to these four peptide hormones for 1, 2, and 3days after the initial decrease in number of the small-cell lung cancercells within the first 24 hours. There was no significant decrease insmall-cell lung cancer number secondary to BNP or CNP (each at 1 μM) at48, 72, or 96 hours (FIG. 7).

The decrease in cancer cell number by ANP, LANP, kaliuretic peptide, andcGMP were significant at P<0.05 while the decrease secondary to thevessel dilator was significant at P<0.001 at each time-point comparedwith placebo when evaluated by repeated analysis of variance (ANOVA).There was no significant decrease in small-cell lung cancer cell numberwith either BNP or CNP when evaluated by repeated ANOVA.

Dose-Response Studies

As BNP and CNP had no effects at 1 μM, the next question asked waswhether they might have effects at 10- and 100-fold higherconcentrations. When evaluated at 10-fold higher concentrations (i.e. 10mM) BNP and CNP decreased the number of human small-cell lung cancercells 2% and 9%, respectively (nonsignificant) (FIG. 8). Increasing BNPand CNP concentrations 100-fold to 100 μM resulted in BNP decreasing thenumber of cancer cells 1% (not significant), but CNP at thisconcentration decreased the number of cells by 39% (P<0.05; FIG. 8).When CNP's effects were evaluated over time at these higherconcentrations, CNP at 10 μM decreased the number of cancer cells at 24,48, 72, and 96 hours 9%, 9%, 9% and 9%, respectively (nonsignificant).At 100 μM, CNP, during this same time period, decreased the number ofsmall-cell lung cancer cells 39%, 39%, 39%, and 38%, respectively(P<0.05).

Neither BNP or CNP had any significant effects on the small-cell lungcancer cell number at their 10-fold higher concentrations (i.e. 10 μM).At a 100-fold higher concentration (i.e. 100 μM), BNP still had noeffect on the small-cell lung cancer cell number, but CNP did cause adecrease in cell number which was significant (P<0.05) when evaluated byANOVA.

Similar dose-response curves revealed that the vessel dilator thatdecreased the number of human small-cell lung cancer cells 63% at 1 μMfurther decreased the number of cancer 73% at 10 μM and 92% at 100 μM(FIG. 9).

At concentrations of 1 μM and higher, the decrease in human small-celllung cancer cells was significant at P<0.001 when evaluated by ANOVA.Comparison of the effects of the different concentrations of the vesseldilator revealed that a significantly (P<0.05) greater decrease (92% incancer cell number) was observed at the 100-μM concentration comparedwith the 1-μM concentration (63% decrease in cancer cell number within24 hours) secondary to the vessel dilator when evaluated by a pairedt-test.

At a 10-μM concentration of the vessel dilator the decrease in humancancer cells at 48, 72, and 96 hours was identical to the decrease at 24hours. When the vessel dilator's effects were examined at 100 μM at 24,48, 72, and 96 hours the decrease in the number of cancer cells was 92%,92%, 92%, and 93% (P<0.001), respectively (FIG. 10).

The respective decreases in the human small-cell lung cancer cell numberat each time point were significant at P<0.001 compared with untreatedhuman cancer cells when evaluated by ANOVA. Thus, the vessel dilator at100 μM eliminated almost all the human small-cell lung cancer cellswithin 24 hours (i.e. there were only 6±0.5 cells left), and theseeffects were sustained with no proliferation of the cancer cells in the3 days after this very significant (P<0.001) decrease in the number ofhuman cancer cells.

Cyclic GMP Decreases Small-Cell Lung Cancer Cell Number Cyclic GMP at 1mM decreased the number of human small-cell lung cancer cells by 31% at24 hours (FIG. 6). The decrease in the number of human small-cellcarcinoma cells at 48, 72, and 96 hours was 40%, 35%, and 32%,respectively (P<0.05 at all time points compared with control) (FIG. 7).

Inhibition of DNA Synthesis by Four Peptide Hormones

To help determine the mechanism of the small-cell lung carcinoma cells'decrease in number and decreased cellular proliferation by the abovefour peptide hormones, the inventors next investigated whether theireffects were owing to an inhibition of DNA synthesis, as they have beendemonstrated to decrease DNA synthesis in pancreatic and breastadenocarcinoma cells. Vessel dilator, LANP, kaliuretic peptide and ANPeach at their 1-μM concentrations inhibited DNA synthesis when incubatedwith human small-cell lung carcinoma cells for 24 hours by 82%, 75%, 68%and 78%, respectively (P<0.001 for each) (FIG. 8). There was nosignificant decrease in DNA synthesis at 1 μM in the small-cell lungcancer cells secondary to BNP or CNP (FIG. 8).

Cyclic GMP Inhibits DNA Synthesis in Small-Cell Lung Cancer Cells

To help define the mechanism(s) for these peptide hormone's ability todecrease DNA synthesis, one of the known mediators of these peptidesbiologic effects, i.e. cyclic GMP, was investigated to determine whetherit could inhibit DNA synthesis in these same small-cell lung cancercells. 8-bromo cyclic GMP decreased DNA synthesis in small-cell lungcarcinoma cells by 50% (P<0.01) at its 1-μM concentration (FIG. 11).

This inhibition of DNA synthesis is illustrated as the percent of DNAsynthesis occurring with the respective peptide hormones, each at 1 μM,vs. the amount of DNA synthesis without the addition of any of thesepeptide hormones. The amount of inhibition of DNA synthesis by each ofthese peptide hormones was significant at P<0.001 when evaluated byrepeated analysis of variance (ANOVA). 8-bromo cyclic GMP at its 1-μMconcentration inhibited DNA synthesis by 54% in the small-cell lungcancer cells (P<0.01). BNP and CNP, each at 1 μM, had no significanteffect on DNA synthesis when evaluated by repeated ANOVA.

NPR-A and -C Receptors are Present in Small-Cell Lung Cancer Cells

Small-cell lung cancer cell lines have been reported to have NPR-Areceptors [35] but natriuretic receptors have never been evaluated inthe small-cell lung cancer line utilized in the present investigation.It has never previously been investigated whether small-cell lungcarcinomas have NPR-C receptors. When the small-cell lung carcinomacells were evaluated by Western blots, the NPRA- and C-receptors weredemonstrated to be present (FIG. 12).

Western blot analysis, FIG. 12, with 1:4000 dilution of A035 polyclonalantibody directed against the COOH terminus of the natriuretic peptideA-receptor (NPRA) and 1:1000 dilution of antibody to the NPR-C receptor.The upper graph demonstrates the positive rat lung control (PANC) forthe NPR-A receptor and the NPR-A receptor in the small-cell lung cancer(SHP-77). The lower graph demonstrates the NPR-C receptor at66-kiloDaltons (kDa) in the human small-cell lung cancer cells (SHP-77)as well as in the positive control (left panel). Beta-galactosidase (130kDa) and BSA (70 kDa) were used in addition to BIO RAD Precision PlusProtein Dual Colour standards to identify the bands corresponding to theNPR-A and NPR-C receptors, respectively. Re-probing with Beta-Actin wasused as a loading control.

Vessel dilator, LANP, kaliuretic peptide and ANP (at 1 μM) and CNP (at100 μM) significantly decrease the number of human small-cell lungcancer cells within 24 hours and inhibit their proliferation for atleast 96 hours. Their mechanism of doing so involves inhibition of DNAsynthesis mediated in part by cyclic GMP.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Furthermore, as used herein, the phrase“pharmaceutically acceptable carrier” means any of the standardpharmaceutically acceptable carriers. The pharmaceutically acceptablecarrier can include diluents, adjuvants, and vehicles, as well asimplant carriers, and inert, non-toxic solid or liquid fillers,diluents, or encapsulating material that does not react with the activeingredients of the invention. Examples include, but are not limited to,phosphate buffered saline, physiological saline, water, and emulsions,such as oil/water emulsions. The carrier can be a solvent or dispersingmedium containing, for example, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils. Formulations are described in anumber of sources that are well known and readily available to thoseskilled in the art. For example, Remington's Pharmaceutical Sciences(Martin E W [1995] Easton Pa., Mack Publishing Company, 19^(th) ed.)describes formulations which can be used in connection with the subjectinvention. Formulations suitable for parenteral administration include,for example, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question. The pharmaceutical composition can beadapted for various forms of administration, including but not limitedto oral, subcutaneous, or intravenous administration. Administration canbe continuous or at distinct intervals as can be determined by a personskilled in the art.

The administration of the ANP prohormone compounds are administered anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight, and other factors known to medical practitioners.

A therapeutically effective amount of each respective peptide hormone,or any combination thereof, is that amount necessary to provide atherapeutically effective amount of the corresponding procyanidin invivo. The amount of prohormone must be effective to achieve a response.In accordance with the present invention, a suitable dose size is a dosethat is capable of preventing or alleviating (reducing or eliminating) asymptom in a patient when administered one or more times over a suitabletime period. One of skill in the art can readily determine appropriatesingle dose sizes for systemic administration based on the size of asubject and the route of administration.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween. Now that theinvention has been described,

1. A method of inhibiting the growth of cancer cells comprising the stepof co-administering, to at least one target cell, an effective amount ofa combination of peptide hormones derived from the atrial natriureticpeptide prohormone.